Method of treating dry eye disease with purinergic receptor agonists

ABSTRACT

A method and preparation for the stimulation of tear secretion in a subject in need of such treatment is disclosed. The method comprises administering to the ocular surfaces of the subject a purinergic receptor agonist such as uridine 5′-triphosphate (UTP), dinucleotides, cytidine 5′-triphosphate (CTP), adenosine 5′-triphosphate (ATP), or their therapeutically useful analogs and derivatives, in an amount effective to stimulate tear fluid secretion and enhance drainage of the lacrimal system. Pharmaceutical formulations and methods of making the same are also disclosed. Methods of administering the same would include: topical administration via a liquid, gel, cream, or as part of a contact lens or selective release membrane; or systemic administration via nasal drops or spray, inhalation by nebulizer or other device, oral form (liquid or pill), injectable, intra-operative instillation or suppository form.

This application is a continuation of U.S. application Ser. No.11/114,957, filed Apr. 25, 2005; now U.S. Pat. No. 7,235,535 which is adivisional of U.S. application Ser. No. 10/041,826, filed Jan. 3, 2002,now U.S. Pat. No. 6,916,794; which is a continuation of U.S. applicationSer. No. 09/171,169, filed Oct. 14, 1998, now U.S. Pat. No. 6,887,858;which was the National Stage of International Application No.PCT/US98/02701, filed Feb. 6, 1998, published Aug. 13, 1998 under PCTArticle 21(2) in English; and was a continuation-in-part of U.S.application Ser. No. 08/797,472 filed Feb. 6, 1997, now U.S. Pat. No.5,900,407. The contents of all the above applications are incorporatedherein by reference.

TECHNICAL FIELD

This invention relates to a method of regulating secretions in andaround the eye of a patient by administering purinergic receptoragonists such as certain uridine, adenine, or cytidine triphosphates aswell as other nucleoside phosphate compounds.

This invention also relates to a method of enhancing drainage of thelacrimal system by administering a pharmacologic agent that enhancesmucociliary clearance of the nasolacrimal duct of a mammal. These agentsinclude certain uridine, adenine and cytidine triphosphates as well asother nucleoside phosphate compounds.

BACKGROUND OF THE INVENTION

There are many situations where it is therapeutically desirable toincrease the amount of tear fluid produced by the eye. Dry eye diseaseis the general term for indications produced by abnormalities of theprecorneal tear film characterized by a decrease in tear production oran increase in tear film evaporation, together with the ocular surfacedisease that results. Approximately 38 million Americans are affectedwith some type of dry eye disorder. Among the indications that arereferred to by the general term “dry eye disease” are:keratoconjunctivitis sicca (KCS), age-related dry eye, Stevens-Johnsonsyndrome, Sjogren's syndrome, ocular cicatrical pemphigoid, blepharitis,corneal injury, infection, Riley-Day syndrome, congenital alacrima,nutritional disorders or deficiencies (including vitamins),pharmacologic side effects, eye stress and glandular and tissuedestruction, environmental exposure to smog, smoke, excessively dry air,airborne particulates, autoimmune and other immunodeficient disorders,and comatose patients rendered unable to blink. The present inventionmay also be useful as a wash or irrigation solution in consciousindividuals, during surgery or to maintain comatose patients or thosewho cannot blink due to neuromuscular blockade or loss of the eyelids.

A healthy precorneal tear film has several important functions: 1) toprotect the cornea from desiccation; 2) to aid in the immune response toinfections; 3) to enhance oxygen permeation into the cornea; 4) to allowgliding movement of the eyeball and eyelids; and 5) to help maintain theocular pressure through osmosis. There are two structures responsiblefor maintaining the properties of the tear film—the lacrimal glands andthe conjunctiva (the mucous membrane which surrounds part of the eyeballand inner eyelids). These structures maintain the tear film viaregulation of water and electrolyte transport and via mucin release bygoblet cells.

The progression of dry eye disease is characterized by four main“milestones.” The first milestone is a decrease in tear production. Inrabbit models, this decrease in tear production has been shown tocorrelate with an increase in tear osmolarity. The second milestone is aloss of mucous-containing conjunctival goblet cells. This decrease ingoblet cell density becomes evident several weeks after the onset ofdecreased tear production. The third milestone in the progression of dryeye disease occurs about 1 year later when desquamation of the cornealepithelium is observed. The fourth and last milestone of the disease isa destabilization of the cornea-tear interface (J. Gilbard, CLAO Journal22(2), 141-45 (1996)).

Currently, the pharmaceutical treatment of dry eye disease is mostlylimited to administration of artificial tears (saline solution) totemporarily rehydrate the eyes. However, relief is short-lived andfrequent dosing is necessary. In addition, artificial tears often havecontraindications and incompatibility with soft contact lenses (M. Lemp,Cornea 9(1), S48-550 (1990)). The use of phosphodiesterase inhibitors,such as 3-isobutyl-1-methylxanthine (IBMX) to stimulate tear secretionis disclosed in U.S. Pat. No. 4,753,945 (applicant intends this and allother patent references to be incorporated herein). The effectiveness ofthese phosphodiesterase inhibitors is currently being investigated (J.Gilbard, et al., Arch. Ophthal, 112, 1614-16 (1994) and 109, 672-76(1991); idem, Inv. Ophthal. Vis. Sci. 31, 1381-88 (1990)). Stimulationof tear secretion by topical application of melanocyte stimulatinghormones is described in U.S. Pat. No. 4,868,154.

There are many situations where it is therapeutically desirable toincrease drainage of the lacrimal system. The lacrimal system has twofunctioning components: the secretory part, which produces tears, andthe excretory part, which drains the tears into the nose. When thelacrimal drainage system is not functioning properly the result can beexcessive tearing (epiphora), mucopurulent discharge, and recurrentdacryocystitis (C. Shermataro, et. al., JAOA, 94, 229 (1994)). In fact,tearing is one of the most common complaints that brings a patient tothe ophthalmologist's office (S. T. Conway, Ophthal. Plas. Reconstr.Surg., 10, 185 (1994)).

The most common malfunction of the lacrimal drainage system isnasolacrimal duct obstruction, which results in stasis of tears in thelacrimal sac. The accumulation of fluid and mucus results in tearing andexpulsion of mucopurulent material, causing the eyelids to be “stucktogether” on awakening in the morning. The lack of clearance of the tearfluid also leads to inflammation and chronic infection of the lacrimalsac and ducts (K. J. Hyde, et. al., Ophthal., 95, 1447 (1988); J. A.Blicker, et. al., Ophthal. Plas. Reconstr. Surg., 9, 43 (1993); J. A.Mauriello Jr., et. al., Ophthal. Plast. Reconstr. Surg., 8, 13 (1992)).

Nasolacrimal duct obstruction can be divided into two etiologic classes:primary acquired nasolacrimal duct obstruction (PANDO), which ischaracterized by hyperplasia and fibrosis of the mucosal epithelium, andsecondary acquired nasolacrimal duct obstruction (SANDO), which iscaused by cancer, inflammation, infection, trauma and mechanicalproblems (G. B. Bartley, Ophthal. Plast. Reconstr. Surg., 8, 237(1992)). An occluded nasolacrimal duct is more common in middle-agedwomen and infants. In fact, up to 20% of all infants are affected bynasolacrimal duct obstruction with most of them becoming symptom free bytheir first birthday (J. D. H. Young, et. al., Eye, 10, 485 (1996)).

Current treatments for nasolacrimal duct obstruction are mostly invasiveor surgical procedures that vary in aggressiveness. Intervention cantake the form of probing the duct with a fine catheter; however, this isa difficult and delicate procedure that requires special training andequipment (J. Kassoff, et. al., Arch. Ophthal., 113, 1168 (1995); J. D.Griffiths, U.S. Pat. No. 4,921,485 (1990) and U.S. Pat. No. 5,062,831(1991); B. B. Becker, et. al., U.S. Pat. No. 5,021,043 (1991) and U.S.Pat. No. 5,169,386 (1992)). In some cases silastic intubation of thenasolacrimal duct increases drainage of tears through the nasolacrimalduct (R. K. Dortzbach, et. al., Amer. J. Ophthal., 94, 585 (1982); H.Al-Hussain, et. al., Ophthal. Plas. Reconstr. Surg., 9, 32 (1993); J. S.Crawford, et. al. U.S. Pat. No. 4,380,239 (1983); W. L. Ector, Jr., U.S.Pat. No. 4,658,816 (1987)). A more aggressive procedure is adacryocystorhinostomy which surgically creates a new drainage path abovethe sight of obstruction allowing continuity between the lacrimal sacand the nasal cavity (J. V. Linberg, et. al., Ophthal., 93, 1055 (1986);K. J. Tarbert, Ophthal., 102, 1065 (1995); F. E. O'Donnell, Jr., U.S.Pat. No. 5,345,948 (1994)). External massage of the nasolacrimal ducthas also been shown to increase tear transit times through thenasolacrimal duct (J. A. Foster, et. al., Ophthal. Plas. Reconstr.Surg., 12, 32 (1996)).

Thus, as a result of the ineffectiveness and inconvenience of currenttherapies, medical researchers have sought to develop alternatives forthe treatment of dry eye disorders and nasolacrimal duct disorders. Ithas been shown that uridine 5′-triphosphate (UTP) and adenine5′-triphosphate (ATP) are potent agonists of P2Y₂ purinergic receptorsfound on the surface of human airway epithelium. Activation of theseP2Y₂ purinergic receptors induces chloride and water secretion, helpinghydrate the airway surface secretions. Use of UTP and ATP for thepurpose of treating pulmonary disorders characterized by the retentionof lung mucus secretions is described in U.S. Pat. No. 5,292,498.Because of the demonstrated ability of UTP to increase hydration ofairway epithelial secretions, applicants were motivated to investigatewhether UTP and other P2Y₂ and P2Y₄ purinergic receptor agonists couldalso stimulate hydration of ocular epithelia. It had previously beenshown that P2 type purinergic receptors in rat and mouse lacrimal acinarcells responded to extracellular ATP by increasing intracellular calcium(I. Sasaki, et al., Febs Lett. 264, 130-34 (1990); idem, J. Physiol.447, 103-18 (1992); P. Vincent, J. Physiol. 449, 313-31 (1992); J.Gromada, et al., Eur J. Physiol. 429, 578 (1995); V. Lee, et al. Inv.Ophthal. Vis. Sci. 38(4)(1997) abstract). Applicant has discovered thattear secretion can be stimulated from lacrimal accessory tissues viaP₂Y₂ and/or P2Y₄ purinergic receptor-mediated mechanisms similar tothose which hydrate airway epithelia. Applicant has also discovered thatstimulators of mucociliary clearance when applied topically to the eyeor injected into the nasolacrimal drainage system increases the flow oftears through the nasolacrimal duct and hence relieves the symptomsassociated with nasolacrimal duct obstruction. UTP and other purinergicreceptor agonists, administered topically or systemically, provide anovel method of treating dry eye disorders and nasolacrimal ductobstruction.

SUMMARY OF THE INVENTION

A method of stimulating tear secretion in a subject in need of suchtreatment is disclosed. The method of the present invention may be usedto increase tear production for any reason, including, but not limitedto, treatment of dry eye disease. Dry eye disease is defined to include:keratoconjunctivitis sicca (KCS), age-related dry eye, Stevens-Johnsonsyndrome, Sjogren's syndrome, ocular cicatrical pemphigoid, blepharitis,corneal injury, infection, Riley-Day syndrome, congenital alacrima,nutritional disorders or deficiencies (including vitamin), pharmacologicside effects, eye stress and glandular and tissue destruction,environmental exposure to smog, smoke, excessively dry air, airborneparticulates, autoimmune and other immunodeficient disorders, andcomatose patients rendered unable to blink. The present invention mayalso be useful as a wash or irrigation solution in consciousindividuals, during surgery or to maintain comatose patients or thosewho cannot blink due to neuromuscular blockade, muscle or nerve damage,or loss of the eyelids. The compound uridine triphosphate (UTP) wasfound to be a potent agonist of P2Y₂ and P2Y₄ purinergic receptors inlacrimal tissue preparations. Furthermore, an in vivo example inaccordance with the invention is conducted on an animal (rabbit) modelof dry eye disease.

A method of enhancing drainage of the lacrimal system in a subject inneed of such treatment is also disclosed. The method of this aspect ofthe invention may be used to enhance clearance of the nasolacrimal ductfor any reason, including, but not limited to, treatment of nasolacrimalduct obstruction. Nasolacrimal duct obstruction is defined to includeboth primary and secondary acquired nasolacrimal duct obstruction andpediatric nasolacrimal duct obstruction. The present invention may alsobe useful as a nasolacrimal wash or irrigation solution in consciousindividuals or during nasolacrimal duct surgery or intubation. Thecompounds disclosed herein may also be used in conjunction withmucolytic agents, such as DNAse, acetylcysteine and bromhexine.

The method of the present invention comprises topically administering aliquid or gel suspension of P2Y₂ and/or P2Y₄ purinergic receptoragonists selected from the group consisting of uridine triphosphate(UTP) and its analogs, P¹,P⁴-di(uridine-5′-) tetraphosphate (U₂P₄) andits analogs, cytidine 5′-triphosphate (CTP) and its analogs, andadenosine 5′-triphosphate (ATP), with the compound of UTP, U₂P₄, CTP orATP administered in an amount effective to stimulate tear secretion orenchance clearance of nasolacrimal ducts.

A second aspect of the present invention is the use of a compound offormula I-IV for the manufacture of a medicament for carrying out atherapeutic method of treatment as given above.

A third aspect of the present invention is a pharmaceutical compositioncomprising a compound of Formula I, II, III or IV, in a pharmaceuticalcarrier in an amount effective to stimulate tear production or toenhance clearance of nasolacrimal ducts in a subject in need of suchtreatment.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the number of mechanical stimuli necessary to induce ablinking reflex is recorded after 5 instillations of 5.0% U₂P₄ isdepicted. in FIG. 1. When compared to saline (left eye), U₂P₄ showed nocorneal anesthesia effects.

FIG. 2 shows the tear secretion effects for 60 minutes after a singledose of U₂P₄ at three concentrations in rabbit eyes are shown in FIG. 2.All three U₂P₄ concentrations increase tear secretion as compared tosaline control. The data are shown as the mean of eight animals.

DETAILED DESCRIPTION OF THE INVENTION

The method of the present invention may be used to increase tearproduction for any reason, including, but not limited to, treatment ofdry eye disease. Dry eye disease is defined to include:keratoconjunctivitis sicca (KCS), age-related dry eye, Stevens-Johnsonsyndrome, Sjogren's syndrome, ocular cicatrical pemphigoid, blepharitis,corneal injury, infection, Riley-Day syndrome, congenital alacrima,nutritional disorders or deficiencies (including vitamin), pharmacologicside effects, eye stress and glandular and tissue destruction,environmental exposure to smog, smoke, excessively dry air, airborneparticulates, autoimmune and other immunodeficient disorders, andcomatose patients rendered unable to blink. The present invention mayalso be useful as a wash or irrigation solution in consciousindividuals, during surgery or to maintain comatose patients or thosewho cannot blink due to muscle or nerve damage, neuromuscular blockadeor loss of the eyelids.

A method of enhancing drainage of the lacrimal system in a subject inneed of such treatment is also disclosed. The method of this aspect ofthe invention may be used to enhance clearance of the nasolacrimal ductfor any reason, including, but not limited to, treatment of nasolacrimalduct obstruction. Nasolacrimal duct obstruction is defined to includeboth primary and secondary acquired nasolacrimal duct obstruction andpediatric nasolacrimal duct obstruction. The present invention may alsobe useful as a nasolacrimal wash or irrigation solution in consciousindividuals or during nasolacrimal duct surgery or intubation. Thecompounds disclosed herein may also be used in conjunction withmucolytic agents, such as DNAse, acetylcysteine and bromhexine.

Applicant has discovered that uridine 5′-triphosphate (UTP) is a potentagonist for purinergic receptors found in lacrimal gland andconjunctival preparations. The method of the present invention is animprovement upon the current most commonly used treatment of dry eyedisease—artificial tears (i.e., saline solution) because UTP stimulatesa patient's own tear production and secretion, which maintain naturalprotective and lubricant characteristics. Furthermore, the method of thepresent invention may be useful even where lacrimal glands aredysfunctional or absent. In addition, the method of the presentinvention may be useful in enhancing clearance of obstructednasolacrimal ducts.

The present invention is concerned primarily with the treatment of humansubjects, but may also be employed for the treatment of other mammaliansubjects, such as dogs and cats, for veterinary purposes.

The term “uridine triphosphate,” as used herein, includes thepharmaceutically acceptable salts thereof, such as (but not limited to)an alkali metal salt such as sodium or potassium; an alkaline earthmetal salt such as magnesium or calcium; or an ammonium or tetraalkylammonium salt, i.e., NX₄ ⁺ (wherein X is C₁₋₄ alkyl). Pharmaceuticallyacceptable salts are salts that retain the desired biological activityof the parent compound and do not impart undesired toxicologicaleffects.

The method of the present invention comprises topically administering aliquid or gel suspension of P2Y₂ and/or P2Y₄ purinergic receptoragonists selected from the group consisting of general Formula I, i.e.,uridine triphosphate [UTP] and its analogs, general Formula II, i.e.,P¹P⁴-di(uridine-5′) tetraphosphate [U₂P₄] and its analogs, generalFormula III, i.e., cytidine 5′-triphosphate [CTP] and its analogs, andgeneral Formula IV, i.e., adenosine 5′-triphosphate [ATP] and itsanalogs, with the particles of Formula I, II, III or IV administered inan amount effective to stimulate tear secretion or to enhance clearanceof nasolacrimal duct obstruction.

Previously described dinucleotides are listed in Table I, along withtheir corresponding literature references.

TABLE I DINUCLEOTIDES IN THE LITERATURE (numbers in parenthesescorrespond to references that follow) Np₂N Np₂N′ Np₃N Np₃N′ Np₄N Np₄N′Ap₂A (4, 1) Ap₂NAD (6) Up₃U (1) Ap₃T (20) Up₄U (2, 3) Ap₄U (3) Gp₂G(5, 1) Ap₂TAD (6) Ap₃A (1, 4, 29) m⁷Gp₃G (5) Ap₄A(1, 4, 29) Ap₄C (3)m⁷Gp₂m⁷G (5) Ap₂C-NAD (6) Xp₃X (1) m^(2,2,7)Gp₃G (5) Cp₄C (3) Ap₄G (3)Ap₂C-PAD (6) m⁷Gp₃m⁷G (5) m^(2,7)Gp₃G (5) Gp₄G (1, 5) Gp₄U (3) Ap₂BAD(6) Gp₃G (1) Xp₄X (1) Gp₄C (3) m⁷Gp₂G (5) Dp₄D (15) Up₄C (3) Up₂U (43)eAp₄eA (7) Ap₄T (20) m⁷Gp₄m⁷G (5) m⁷Gp₄G (5) m^(2,7)Gp₄G (5)m^(2,2,7)Gp₄G (5) AppZppA DppZppD ApZppZpA ApSpZpSpA Z Z Z Z CH₂ (8) CH₂(15) CH₂ (8) CHF (8) CH₂CH₂ (8) CH₂CH₂ (15) CH₂CH₂ (8) CF₂ (8) CHF (8)CHF (15) CHF (8) O (8) CF₂ (8) CF₂ (15) CF₂ (8) CHCl (8) CHCl (15) CHCl(8) CCl₂ (8) CCl₂ (15) CCl₂ (8) A = Adenosine U = Uridine G = GuanosineT = Thymidine X = Xanthosine TAD = Tiazofurin BAD = Benzamide riboside D= 2,6-Diaminopurine eA = Ethenoadenosine m⁷G = 7-Methylguanosinem^(2,7)G = 2,7-Dimethylguanosine m^(2,2,7)G = 2,2,7-TrimethylguanosineNAD = nicotinamide riboside C-NAD = C-nicotinamide riboside C-PAD =C-picolinamide riboside N = Nucleoside (1) M. A. G. Sillero et al., Eur.J. Biochem., 76, 331 (1977) (2) C. G. Vallejo et al., Biochim. Biophys.Acta, 483, 304 (1976) (3) H. Coste et al., J. Biol. Chem., 262, 12096(1987) (4) K. E. Ng et al., Nucleic Acid Res., 15, 3573 (1987) (5) J.Stepinski et al., Nucleosides & Nucleotides, 14, 717 (1995) (6) A.Zatorski et al., J. Med. Chem., 39, 2422 (1996) (7) P. Rotilan et al.,FEBS, 280, 371 (1991) (8) P. C. Zamecnik et al., Proc. Natl. Acad. Sci.,89, 2370 (1992) (9) J. Walker et al., Biochemistry, 32, 14009 (1993)(10) R. H. Hiderman et al., J. Biol. Chem., 266, 6915 (1991) (11) J.Luthje et al., Eur. J. Biochem., 173, 241 (1988) (12) R. H. Silverman etal., Microbiological Rev., 43, 27 (1979) (13) C. D. Lobaton et al., Eur.J. Biochem., 50, 495 (1975) (14) G. Lowe et al., Nucleosides &Nucleotides, 10, 181 (1991) (15) G. M. Blackburn et al., Nucleosides &Nucleotides, 10, 549 (1991) (16) J. C. Baker et al., Mutation Res., 208,87 (1988) (17) G. Klein et al., Biochemistry, 27, 1897 (1988) (18) E.Castro et al., Br. J. Pharmacol., 100, 360 (1990) (19) D. R. Elmaleh etal., Proc. Natl. Acad. Sci., 81, 918 (1984) (20) R. Bone et al., J.Biol. Chem., 261, 16410 (1986) (21) Fed. Amer. Soc. Exper. Bio., Abstr.Part I, no. 1878 (1991) (22) M. T. Miras-Portugal et al., Ann. NY Acad.Sci., 603, 523 (1990) (23) A. Guranowski et al., Biochemistry, 27, 2959(1988) (24) F. Grummt et al., Plant Mol. Bio., 2, 41 (1983) (25) A. G.McLennan et al., Nucleic Acid Res., 12, 1609 (1984) (26) P. Zamecnik etal., Analytical Biochem., 134, 1 (1983) (27) E. Rapaport et al., Proc.Natl. Acad. Sci., 78, 838 (1981) (28) T. Kimura et al., Biol. Pharm.Bull., 18, 1556 (1995) (29) E. Schulze-Lohoff et al., Hypertension, 26,899 (1995) (30) B. K. Kim et al., Proc. Natl. Acad. Sci., 89, 11056(1992) (31) P. C. Zamecnik et al., Proc. Natl. Acad. Sci., 89, 2370(1992) (32) H. Morii et al., Eur. J. Biochem., 205, 979 (1992) (33) E.Castro et al., Pflugers Arch., 426, 524 (1994) (34) H. Schluter et al.,Nature, 367, 186 (1994) (35) E. Castro et al., Br. J. Pharmacol., 206,833 (1992) (36) T. Casillas et al., Biochemistry, 32, 14203 (1993) (37)J. Pintor et al., J. Neurochem., 64, 670 (1995) (38) E. Castro et al.,J. Biol. Chem., 270, 5098 (1995) (39) V. A. Panchenko et al.,Neuroscience, 70, 353 (1996) (40) E. Castro et al., Br. J. Pharmacol.,100, 360 (1990) (41) J. Pintor et al., Gen. Pharmac., 26, 229 (1995)(42) J. Pintor et al., Br. J. Phamacol., 115, 895 (1995) (43) A.Kanavarioti et al., Tett. Lett., 32, 6065 (1991)

Active Compounds of the Invention

UTP and its analogs are depicted in general Formula I:

wherein:

X₁, X₂ and X₃ are each independently either O⁻ or S⁻. Preferably, X₂ andX₃ are O⁻.

R₁ is O, imido, methylene or dihalomethylene (e.g., dichloromethylene ordifluoromethylene). Preferably, R₁ is oxygen or imido.

R₂ is H or Br. Preferably, R₂ is H. Particularly preferred compounds ofFormula I are uridine 5′-triphosphate (UTP) and uridine5′-O-(3-thiotriphosphate) (UTPγS).

A dinucleotide is depicted by the general Formula II:

wherein:

X is oxygen, imido, methylene or difluoromethylene;

n=0 or 1;

m=0 or 1;

n+m=0, 1 or 2; and

B and B′ are each independently a purine residue or a pyrimidine residuelinked through the 9- or 1-position, respectively. In the instance whereB and B′ are uracil, attached at the N-1 position to the ribosyl moiety,then the total of m+n may equal 3 or 4 when X is oxygen. The ribosylmoieties are in the D-configuration, as shown, but may be L-, or D- andL-. The D-configuration is preferred.

B and B′ are each independently a purine residue, as in Formula IIa, ora pyrimidine residue, as in Formula IIb, linked through the 9- or1-position, respectively. In the instance where B and B′ are uracil,attached at the N-1 position to the ribosyl moiety, then the total ofm+n may equal 3 or 4 when X is oxygen. The ribosyl moieties are in theD-configuration, as shown, but may be L-, or D- and L-. TheD-configuration is preferred.

The substituted derivatives of adenine include adenine 1-oxide; 1,N6-(4-or 5-substituted etheno) adenine; 6-substituted adenine; or8-substituted aminoadenine, where R₃ is H or NHR₁, where R₁ of the 6- or8-HNR′ groups are chosen from among: hydrogen, arylalkyl (C₁₋₆) groupswith the aryl moiety optionally functionalized as described below;alkyl; and alkyl groups with functional groups therein, such as:([6-aminohexyl]carbamoylmethyl)-, and ω-acylated-amino(hydroxy, thioland carboxy) derivatives where the acyl group is chosen from among, butnot limited to, acetyl, trifluroroacetyl, benzoyl, substituted-benzoyl,etc., or the carboxylic moiety is present as its ester or amidederivative, for example, the ethyl or methyl ester or its methyl, ethylor benzamido derivative. The ω-amino(hydroxy, thiol) moiety may bealkylated with a C₁₋₄ alkyl group.

Likewise, B or B′, or both may be a pyrimidine with the general formulaof FIG. IIb, linked through the 1-position:

wherein:

R₄ is hydroxy, mercapto, amino, cyano, aralkoxy, C₁₋₆ alkoxy, C₁₋₆alkylamino and dialkylamino, the alkyl groups optionally linked to forma heterocycle;

R₅ is hydrogen, acyl, C₁₋₆ alkyl, aroyl, C₁₋₅ alkanoyl, benzoyl, orsulphonate;

R₆ is hydroxy, amino, mercapto, alkoxy, aralkoxy, C₁₋₆-alkylthio, C₁₋₅disubstituted amino, triazolyl, alkylamino or dialkylamino, where thealkyl groups are optionally linked to form a heterocycle or linked to N³to form an optionally substituted ring;

R₇ is hydrogen, hydroxy, cyano, nitro, alkenyl with the alkenyl moietyoptionally linked through oxygen to form a ring optionally substitutedon the carbon adjacent to the oxygen with alkyl or aryl groups,substituted alkynyl, halogen, alkyl, substituted alkyl, perhalomethyl(e.g., CF₃), C₂₋₆ alkyl, C₂₋₃ alkenyl, or substituted ethenyl (e.g.,allylamino, bromvinyl and ethyl propenoate, or propenoic acid), C₂₋₃alkynyl or substituted alkynyl; or together R₆-R₇ may form a 5 or6-membered saturated or unsaturated ring bonded through N or O at R₆,such a ring may contain substituents that themselves containfunctionalities; provided that when R₈ is amino or substituted amino, R₇is hydrogen; and

R₈ is hydrogen, alkoxy, arylalkoxy, alkylthio, arylalkylthio,carboxamidomethyl, carboxymethyl, methoxy, methylthio, phenoxy orphenylthio.

In the general structure of FIG. IIb above, the dotted lines in the 2-to 6positions are intended to indicate the presence of single or doublebonds in these positions; the relative positions of the double or singlebonds being determined by whether the R₄, R₆ and R₇ substituents arecapable of keto-enol tautomerism.

In the general structures of FIG. Ia and IIb above, the acyl groupsadvantageously comprise alkanoyl or aroyl groups. The alkyl groupsadvantageously contain 1 to 8 carbon atoms, particularly 1 to 4 carbonatoms optionally substituted by one or more appropriate substituents, asdescribed below. The aryl groups including the aryl moieties of suchgroups as aryloxy are preferably phenyl groups optionally substituted byone or more appropriate substituents, as described below. The abovementioned alkenyl and alkynyl groups advantageously contain 2 to 8carbon atoms, particulary 2 to 6 carbon atoms, e.g., ethenyl or ethynyl,optionally substituted by one or more appropriate substituents asdescribed below. Appropriate substituents on the above-mentioned alkyl,alkenyl, alkynyl, and aryl groups are advantageously selected fromhalogen, hydroxy, C₁₋₄ alkoxy, C₁₋₄ alkyl, C₇₋₁₂ arylalkoxy, carboxy,cyano, nitro, sulfonamido, sulfonate, phophate, sulfonic, amino andsubstituted amino wherein the amino is singly or doubly substituted by aC₁₋₄ alkyl, and when doubly substituted, the alkyl groups optionallybeing linked to form a heterocycle.

ATP and its analogs are depicted by general Formula III:

wherein:

R₁, X₁, X₂ and X₃ are defined as in Formula I.

R₃ and R₄ are H while R₂ is nothing and there is a double bond betweenN-1 and C-6 (adenine), or

R₃ and R₄ are H while R₂ is O and there is a double bond between N-1 andC-6 (adenine 1-oxide), or

R₃, R₄ and R₂ taken together are —CH═CH—, forming a ring from N-6 to N-1with a double bond between N-6 and C-6 (1,N6-ethenoadenine).

CTP and its analogs are depicted by general Formula IV:

wherein:

R₁, X₁, X₂ and X₃ are defined as in Formula I.

R₅ and R₆ are H while R₇ is nothing and there is a double bond betweenN-3 and C-4 (cytosine), or

R₅, R₆ and R₇ taken together are —CH═CH—, forming a ring from N-3 to N-4with a double bond between N-4 and C-4 (3,N⁴-ethenocytosine) optionallysubstituted at the 4- or 5-position of the etheno ring.

For simplicity, Formulae I, II, III and IV herein illustrate the activecompounds in the naturally occurring D-configuration, but the presentinvention also encompasses compounds in the L-configuration, andmixtures of compounds in the D- and L-configurations, unless otherwisespecified. The naturally occurring D-configuration is preferred.

The active compounds of the invention may also be present in the form oftheir pharmaceutically acceptable salts, such as, but not limited to, analkali metal salt such as sodium or potassium; an alkaline earth metalsalt such as manganese, magnesium or calcium; or an ammonium ortetraalkyl ammonium salt, i.e., NX₄ ⁺ (wherein X is C₁₋₄).Pharmaceutically acceptable salts are salts that retain the desiredbiological activity of the parent compound and do not impart undesiredtoxicological effects.

Methods of Administration

The active compounds disclosed herein may be administered to the eyes ofa patient by any suitable means, but are preferably administered byadministering a liquid or gel suspension of the active compound in theform of drops, spray or gel. Alternatively, the active compounds may beapplied to the eye via liposomes. Further, the active compounds may beinfused into the tear film via a pump-catheter system. Anotherembodiment of the present invention involves the active compoundcontained within a continuous or selective-release device, for example,membranes such as, but not limited to, those employed in the Ocusert™System (Alza Corp., Palo Alto, Calif.). As an additional embodiment, theactive compounds can be contained within, carried by, or attached tocontact lenses which are placed on the eye. Another embodiment of thepresent invention involves the active compound contained within a swabor sponge which can be applied to the ocular surface. Another embodimentof the present invention involves the active compound contained within aliquid spray which can be applied to the ocular surface. Anotherembodiment of the present invention involves an injection of the activecompound directly into the lacrimal tissues or onto the eye surface.

The quantity of the active compound included in the topical solution isan amount sufficient to achieve dissolved concentrations of the activecompound on the ocular surface of the subject of from about 10⁻⁷ toabout 10⁻¹ Moles/liter, and more preferably from about 10⁻⁶ to about10⁻¹ Moles/liter, in order to stimulate tear secretion or enhanceclearance of nasolacrimal ducts.

Depending upon the solubility of the particular formulation of activecompound administered, the daily dose to promote tear secretion orenhance clearance of nasolacrimal duct clearance may be divided amongone or several unit dose administrations. The total daily dose for UTP(for example) may range from a concentration of 0.25 mg/ml to 50 mg/ml,depending upon the age and condition of the subject. A currentlypreferred unit dose for UTP is about 1 to 100 milligrams given at aregimen of 2 to 6 administrations per day.

Some compounds of Formula I, III and IV can be made by methods which arewell known to those skilled in the art; some are commercially available,for example, from Sigma Chemical Company, PO Box 14508, St. Louis, Mo.63178. Compounds of Formula II can be made in accordance with knownprocedures, or variations thereof which will be described by: P.Zamecnik, et al., Proc. Natl. Acad. Sci. USA 89, 838-42 (1981); and K.Ng and L. E. Orgel, Nucleic Acids Res. 15(8), 3572-80 (1977).

The topical solution containing the active compound may also contain aphysiologically compatible vehicle, as those skilled in the ophthalmicart can select using conventional criteria. The vehicles may be selectedfrom the known ophthalmic vehicles which include, but are not limitedto, saline solution, water polyethers such as polyethylene glycol,polyvinyls such as polyvinyl alcohol and povidone, cellulose derivativessuch as methylcellulose and hydroxypropyl methylcellulose, petroleumderivatives such as mineral oil and white petrolatum, animal fats suchas lanolin, polymers of acrylic acid such as carboxypolymethylene gel,vegetable fats such as peanut oil and polysaccharides such as dextrans,and glycosaminoglycans such as sodium hyaluronate and salts such assodium chloride and potassium chloride.

In addition to the topical method of administration described above,there are various methods of administering the active compounds of thepresent invention systemically. One such means would involve an aerosolsuspension of respirable particles comprised of the active compound,which the subject inhales. The active compound would be absorbed intothe bloodstream via the lungs or contact the lacrimal tissues vianasolacrimal ducts, and subsequently contact the lacrimal glands in apharmaceutically effective amount. The respirable particles may beliquid or solid, with a particle size sufficiently small to pass throughthe mouth and larynx upon inhalation; in general, particles ranging fromabout 1 to 10 microns, but more preferably 1-5 microns, in size areconsidered respirable.

Another means of systemically administering the active compounds to theeyes of the subject would involve administering a liquid/liquidsuspension in the form of eye drops or eye wash or nasal drops of aliquid formulation, or a nasal spray of respirable particles which thesubject inhales. Liquid pharmaceutical compositions of the activecompound for producing a nasal spray or nasal or eye drops may beprepared by combining the active compound with a suitable vehicle, suchas sterile pyrogen free water or sterile saline by techniques known tothose skilled in the art.

Other means of systemic administration of the active compound wouldinvolve oral administration, in which pharmaceutical compositionscontaining compounds of Formula I, II, III or IV are in the form oftablets, lozenges, aqueous or oily suspensions, dispersible powders orgranules, emulsion, hard or soft capsules, or syrups or elixirs.Compositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavoringagents, coloring agents and preserving agents in order to providepharmaceutically elegant and palatable preparations. Tablets contain theactive ingredient in admixture with nontoxic pharmaceutically acceptableexcipients which are suitable for the manufacture of tablets. Theseexcipients may be, for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example, cornstarch, or alginic acid; binding agents, for example, starch, gelatin oracacia; and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets may be uncoated or they may be coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed. Formulations fororal use may also be presented as hard gelatin capsules wherein theactive ingredient is mixed with an inert solid diluent, for example,calcium carbonate, calcium phosphate or kaolin, or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, for example, peanut oil, liquid paraffin or olive oil.

Additional means of systemic administration of the active compound tothe eyes of the subject would involve a suppository form of the activecompound, such that a therapeutically effective amount of the compoundreaches the eyes via systemic absorption and circulation.

Further means of systemic administration of the active compound wouldinvolve direct intra-operative instillation of a gel, cream, or liquidsuspension form of a therapeutically effective amount of the activecompound.

Those having skill in the art will recognize that the starting materialsmay be varied and additional steps employed to produce compoundsencompassed by the present invention, as demonstrated by the followingexamples. In some cases protection of certain reactive functionalitiesmay be necessary to achieve some of the above transformations. Ingeneral the need for such protecting groups will be apparent to thoseskilled in the art of organic synthesis as well as the conditionsnecessary to attach and remove such groups.

The invention is illustrated further by the following examples which arenot to be construed as limiting the invention in scope or spirit to thespecific procedures described in it. In vivo examples in accordance withthe invention are conducted on rabbits with dry eyes. The dry eyedisorder is created by surgically closing the duct that carries fluidfrom the main lacrimal gland to the tear film and surgically removingthe nictitans and harderian glands. It is recognized by those skilled inthe art that results of ophthalmologic tests carried out on theaforementioned rabbit model have close correlation with humans afflictedwith dry eye disease, and, therefore, the results provide an accurateprediction of therapeutic efficacy in humans.

EXAMPLES Example 1 Stimulation of Mucin Release in Rat ConjunctivalPreparations

Male, Sprague-Dawley rats at 12 weeks of age (Charles RiverLaboratories, Wilmington, Mass.) are sacrificed by intraperitonealinjection of sodium pentobarbital (1300 mg/kg) and a 20 μL drop of 1%lidocaine diluted with tear buffer (106.5 mM NaCl, 26.1 mM NaHCO₃, 18.7mM KCl, 1.0 mM MgCl₂, 0.5 mM NaH₂PO₄, 1.1 mM CaCl₂, and 10 mM HEPES pH7.45) is placed on the ocular surface for 10 min. The inferiorconjunctiva extending from lid to cornea and medial to lateral canthusis removed from each eye and placed on filter paper. Each conjunctiva iscut in half longitudinally and the four resulting pieces are incubatedin keratinocyte growth medium (Clonetics Corp., San Diego, Calif.) withor without agonist for 1 hr at 4° C. in half strength Karnovsky'ssolution (2.5% glutaraldehyde and 2% paraformaldehyde in cacodylatebuffer, pH 7.4), embedded in methacrylate, and sectioned into 3 μmpieces. Six sections from each piece of tissue are stained with AlcianBlue (pH 5) and periodic acid-Schiffs reagent (AB-PAS). The number ofmucin-containing goblet cells in each section is counted in maskedfashion using light microscopy (standard microscope with a squareindexed eye piece reticule) at 160× magnification. In unstimulatedconjunctival tissue culture the goblet cells have defined borders andare intensely stained due to the mucin-containing secretory granules inthe apical portion of each goblet cell. Upon stimulation the mucingranules are released into the medium. The number of quiescientmucin-containing goblet cells per unit area (0.16 mm²) is counted andaveraged. A decrease in the number of mucin-containing goblet cells perunit area indicate an increase in mucin secretion, because the stainingis caused by non-secreted cells. Data are expressed as the meanpercentage of the value from the control (untreated) tissue. The methodof this example is adapted from D. Dartt, et al., Exp. Eye Res., 63, 27(1996), which is incorporated herein by reference.

Example 2 Measurement of Intracellular Calcium

Rat conjunctival cells grown on vitrogen coated coverslips are loadedwith a final concentration of 3 μM Fura-2/AM at 37° C. for 30 min. Thecells are then washed in NaCl ringer and mounted in a chamber formeasurement of fluorescence. To reduce the rate of leakage of Fura-2from the cell into the extracellular space and avoid time-dependentcompartmentalization of the probe, all measurements of [Ca²⁺]_(i) areconducted at 25° C. At this temperature, no vesicular bright spotsindicative of compartmentalization of the probe are observed.

Measurements of [Ca²⁺]_(i) in single rat conjunctival epithelial cellsare obtained with a modular microspectrofluorometer (SPEX Industries,Inc., Edison, N.J.) attached to a Zeiss Axiovert IM 35 microscope. Thesystem is equipped with a xenon lamp, beam splitter, two monochromatorsand a rotating chopper mirror that permit excitation of cellfluorescence at alternating wavelengths of 340 and 380 nm (emission >450nm). The fluorescent signal from a single cell is measured with aphotometer equipped with a pinhole (spot diameter of 3-5 μm) thatexclude signals from adjacent cells.

After agonist is added, the fluorescent signal is quenched by a NaClringer solution containing 1.5×10⁻⁴M digitonin and 10⁻³M MnCl₂. Theremaining signal at each excitation wavelength, equivalent to thebackground fluorescence in non-loaded cells, is subtracted from datafrom Fura-2/AM loaded cells before the ratio (340/380 nm) is taken. The340 nm/380 nm ratio is converted to an actual [Ca²⁺]_(i) measurement byusing the external calibration standards and the formula derived by G.Grynkiewicz, et al. (J. Biol. Chem. 260, 3440-3450 (1985)), used withdual wavelength measurements: [Ca2+]_(i)=K[(R_(x)-R_(o))/(R_(s)-R_(x))], with R_(o) and R_(s) representing theratios at zero Ca²⁺ and saturating Ca²⁺, respectively. R_(x) representsthe experimental ratio. K is K_(d)/(F_(o)/F_(s)), with K_(d)=1.57×10⁻⁷ Mat 25° C. as the effective dissociation constant for Fura-2, and F_(o)and F_(s) represent the fluorescence intensities at 380 nm with zero andsaturating Ca²⁺, respectively. The method of this example is adaptedfrom R. Boucher, et al., U.S. Pat. No. 5,292,498, which is incorporatedherein by reference.

Example 3 Reversing the Onset of Dry Eye Disease in Rabbit Model for KCS

Keratoconjunctivitis sicca (KCS) is created in the right eyes of 8 NewZealand white rabbits by surgically closing the lacrimal gland excretoryduct, and removing the nictitating membrane, nictitans gland andHarderian gland. All rabbits are left untreated for 8 weeks and KCS isconfirmed by measuring elevated tear film osmolarity by taking 0.1-0.4μL tear samples as previously described (J. Gilbard, et al., Ophthalmol.96, 677 (1978)). A 3.0 mmol solution of UTP or analog is prepared in apreserved, isotonic buffer solution. Four of the rabbits are treatedwith 1 drop (10 μL) UTP or analog solution four times a day, excludingweekends. The remaining 4 untreated rabbits serve as controls. Aftertreatment began, 0.1-0.4 μL tear samples are taken from all rabbits forosmolarity measurements on Monday mornings before the first dose. At 20weeks the animals are sacrificed and goblet cell densities are measuredby staining with alcian blue and periodic acid-Schiff's reagent (D.Dartt, et al., Exp. Eye Res. 67, 27 (1996)).

The study is designed to demonstrate that UTP and analogs decreaseelevated tear film osmolarity and increased conjunctival goblet celldensities, thus reversing the onset of ocular surface disease in arabbit model for KCS. The method of this example is adapted from J. P.Gilbard, Arch. Ophthalmol. 112, 1614 (1994), which is incorporatedherein by reference.

Example 4 Acute Ocular Tolerance in Rabbits

U₂P₄ (P¹,P⁴-Di(uridine tetraphosphate), tetrasodium salt) was formulatedas an isotonic aqueous solution and topically administered to the eyesof albino rabbits in a series of experiments to provide a broadindication of the acute ocular safety of U₂P₄. The experiments wereconducted according to GLP guidelines. A modified Draize test was usedto determine whether U₂P₄ is acceptable for administration to the eye.

Healthy, male, adult albino New Zealand rabbits (range 2-2.5 kg) wereused for these studies. Rabbits were obtained from Elevage Scientifiquedes Dombes (Chantillon sur Charlaronne, France). Animals were observeddaily for signs of ill health and only healthy animals with no ocularabnormalities were used for experiments. Animals were housed in standardcages in one room under controlled environmental conditions. Animals hadfree access to food and water throughout the study. The test article forall studies was daily formulated in water and NaCl to make an isotonicsolution.

This experiment was an open trial in which U₂P₄ at a 5.0% concentrationwas delivered by multiple 50 μL instillations (5 times in 20 minutes)into the conjunctival sac of the right eye of three rabbits. Animalswere assigned a clinical ocular safety grade for the conjunctiva, corneaand iris according to a modified Draize scale at 0, 1, 2 and 3 hoursafter the last instillation. The left eye was instilled withphysiological saline and served as a control.

The ocular safety results for U₂P₄ at the 5.0% concentration show onlyone rabbit (both eyes) with slight conjunctival redness (grade 1 on a0-4 scale of increasing severity). All other grades for conjunctiva,cornea and iris for redness, chemosis and watering were zeros (see Table2 in appended report). Results for placebo administration were alsozeros in all cases. U₂P₄ was thus considered safe for administration tothe eye.

Example 5 Corneal Anesthesia Effects in Rabbits

U₂P₄ (P¹,P⁴-Di(uridine 5′-)tetraphosphate), tetrasodium salt) wasformulated as an isotonic aqueous solution and topically administered tothe eyes of albino rabbits to provide a broad indication of the acuteocular safety of U₂P₄. The experiments were conducted according to GLPguidelines.

Healthy, male, adult albino New Zealand rabbits (range 2-2.5 kg) wereused for these studies. Rabbits were obtained from Elevage Scientifiquedes Dombes (Chantillon sur Charlaronne, France). Animals were observeddaily for signs of ill health and only healthy animals with no ocularabnormalities were used for experiments. Animals were housed in standardcages in one room under controlled environmental conditions. Animals hadfree access to food and water throughout the study. The test article forall studies was daily formulated in water and NaCl to make an isotonicsolution.

U₂P₄ at 5.0% concentration was delivered by multiple 50 μL instillations(5 times in 20 minutes) into the conjunctival sac of the right eye ofthree rabbits and the corneal anesthetic effects evaluated with aCochet's esthesiometer at 5, 10, 20, 30, 40, 50 and 60 minutes after thelast instillation. Corneal anesthesia was evaluated by the number ofcorneal mechanical stimuli necessary to induce a blinking reflex. Theleft eye was instilled with physiological saline and served as acontrol.

The safety of U₂P₄ 5.0% solution is demonstrated further, because itshowed no corneal anesthetic effects when administered to the right eyesof albino rabbits (see FIG. 1).

Example 6 Tear Secretion in Rabbits

U₂P₄ (P¹,P⁴-Di(uridine tetraphosphate), tetrasodium salt) was formulatedas an isotonic aqueous solution and topically administered to the eyesof albino rabbits and tear secretion experiments were conducted as ameasure of efficacy in normal rabbits.

Healthy, male, adult albino New Zealand rabbits (range 2-2.5 kg) wereused for these studies. Rabbits were obtained from Elevage Scientifiquedes Dombes (Chantillon sur Charlaronne, France). Animals were observeddaily for signs of ill health and only healthy animals with no ocularabnormalities were used for experiments. Animals were housed in standardcages in one room under controlled environmental conditions. Animals hadfree access to food and water throughout the study. The test article forall studies was daily formulated in water and NaCl to make an isotonicsolution.

U₂P₄ at 0.5%, 5.0% and 8.5% concentrations was instilled (50 μL) 5 timesa day for 14 days into the conjunctival sac of eight rabbits in separategroups. Tear secretion was measured using a Schirmer test strip at 0, 5,15, 30 and 60 minutes after the first and last instillation of the dayon days 1, 7 and 14. The results are compared to separate saline anduntreated control groups.

All three concentrations of U₂P₄ increased tear secretion in rabbit eyesover a 60-minute period as compared to saline control (see FIG. 2).

The invention and the manner and process of making and using it are nowdescribed in such full, clear, concise and exact terms as to enable anyperson skilled in the art to which it pertains, to make and use thesame. It is to be understood that the foregoing describes preferredembodiments of the present invention and that modifications may be madetherein without departing from the spirit or scope of the presentinvention as set forth in the claims. To particularly point out anddistinctly claim the subject matter regarded as invention, the followingclaims conclude this specification.

1. A method of treating dry eye disease, comprising the step ofadministering to the eyes of a subject in need thereof an effectiveamount of a dinucleotide compound as depicted in Formulae II, or apharmaceutically acceptable salt thereof; and whereby said dinucleotidecompound is effective in promoting tear secretion and mucin productionin the eyes in a subject in need of such treatment:

wherein: X is oxygen, imido, methylene or difluoromethylene; n=0 or 1;m=0 or 1; n+m =0, 1 or 2; and B and B′ are each independently a purineresidue, as in Formula IIa, or a pyrimidine residue, as in Formula IIb,linked through the 9- or 1-position, respectively:

wherein: R₃ is NHR₁; R₁ of the 6- or 8-HNR₁ groups is selected from thegroup consisting of hydrogen, arylalkyl (C₁₋₆) groups; and alkyl groupswith functional groups selected from the group consisting of([6-aminohexyl]carbamoylmethyl)-, ω-acylated-(amino, hydroxy, thiol orcarboxy)alkyl(C₂₋₁₀)— and ω-acylated-(amino, hydroxy, thiol or carboxy)derivatives where the acyl group is selected from the group consistingof acetyl, trifluoroacetyl, benzoyl, and substituted-benzoyl; R₂ is O orabsent; or R₁ and R₂ taken together form a substituted 5-membered fusedimidazole ring;

wherein: R₄ is hydroxy, mercapto, amino, cyano, aralkoxy, C₁₋₆ alkoxy,C₁₋₆ alkylamino or dialkylamino, with the alkyl groups optionally linkedto form a heterocycle; R₅ is hydrogen, acyl, C₁₋₆ alkyl, aroyl, C₁₋₅alkanoyl, benzoyl, or sulphonate; R₆ is hydroxy, mercapto, alkoxy,aralkoxy, C₁₋₆-alkylthio, C₁₋₅ disubstituted amino, triazolyl,alkylamino or dialkylamino, where the alkyl groups are optionally linkedto form a heterocycle or linked to N³ to form an optionally substitutedring; R₇ is hydrogen, hydroxy, cyano, nitro, alkenyl with the alkenylmoiety optionally linked through oxygen to form a ring optionallysubstituted on the carbon adjacent to the oxygen with alkyl or arylgroups, halogen, alkyl, substituted alkyl, perhalomethyl, C₂₋₆ alkyl,C₂₋₃ alkenyl, or substituted ethenyl, C₂₋₃ alkynyl or substitutedalkynyl; or together R₆-R₇ form a 5 or 6-membered saturated orunsaturated ring bonded through N or O at R₆, such a ring optionallycontains substituents that themselves contain functionalities; and R₈ ishydrogen, alkoxy, arylalkoxy, alkylthio, arylalkylthio,carboxamidomethyl, carboxymethyl, methoxy, methylthio, phenoxy orphenylthio.
 2. The method according to claim 1, wherein saidadministering is topically administering said compound via a carriervehicle selected from the group consisting of drops of liquid, liquidwash, gels, ointments, sprays and liposomes.
 3. The method according toclaim 2, wherein said topically administering comprises infusing saidcompound to said eyes via a device selected from the group consisting ofa pump-catheter system, a continuous or selective release device, and acontact lens.
 4. The method according to claim 1, wherein saidadministering is systemically administering a liquid or liquidsuspension of said compound via nose drops or nasal spray or nebulizedliquid to oral or nasopharyngeal airways of said subject, such that atherapeutically effective amount of said compound contacts the eyes ofsaid subject via systemic absorption and circulation.
 5. The methodaccording to claim 4, wherein said administering is systemicallyadministering an oral form of said compound, such that a therapeuticallyeffective amount of said compound contacts the eyes of said subject viasystemic absorption and circulation.
 6. The method according to claim 1,wherein said administering is administering an injectable form of saidcompound, such that a therapeutically effective amount of said compoundcontacts the lacrimal tissues of said subject via systemic absorptionand circulation.
 7. The method according to claim 1, wherein saidadministering is administering a suppository form of said compound, suchthat a therapeutically effective amount of said compound contacts thelacrimal tissues of said subject via systemic absorption andcirculation.
 8. The method according to claim 1, wherein saidadministering is administering an intra-operative instillation of a gel,cream, powder, foam, crystals, liposomes, spray or liquid suspensionform of said compound.
 9. The method according to claim 1, wherein saidcompound is administered in an amount sufficient to achieveconcentrations thereof on the ocular surfaces of said subject of fromabout 10⁻⁷ to about 10⁻¹ moles/liter.